Natural Killer Cell-Derived Extracellular Vesicles as a Promising Immunotherapeutic Strategy for Cancer: A Systematic Review

Cancer is the second leading contributor to global deaths caused by non-communicable diseases. The cancer cells are known to interact with the surrounding non-cancerous cells, including the immune cells and stromal cells, within the tumor microenvironment (TME) to modulate the tumor progression, metastasis and resistance. Currently, chemotherapy and radiotherapy are the standard treatments for cancers. However, these treatments cause a significant number of side effects, as they damage both the cancer cells and the actively dividing normal cells indiscriminately. Hence, a new generation of immunotherapy using natural killer (NK) cells, cytotoxic CD8+ T-lymphocytes or macrophages was developed to achieve tumor-specific targeting and circumvent the adverse effects. However, the progression of cell-based immunotherapy is hindered by the combined action of TME and TD-EVs, which render the cancer cells less immunogenic. Recently, there has been an increase in interest in using immune cell derivatives to treat cancers. One of the highly potential immune cell derivatives is the NK cell-derived EVs (NK-EVs). As an acellular product, NK-EVs are resistant to the influence of TME and TD-EVs, and can be designed for “off-the-shelf” use. In this systematic review, we examine the safety and efficacy of NK-EVs to treat various cancers in vitro and in vivo.


Introduction
According to the World Health Organization (WHO), the COVID-19 pandemic has claimed over 6.6 million lives globally [1]. Nonetheless, the incidence of infections and deaths has declined significantly with the availability of vaccines. Contradictorily, solutions for cancer treatment still remain obscure today. In 2021 alone, the WHO recorded 9.3 million global deaths from neoplasms [2]. Chemotherapy and radiotherapy are the standard treatment for cancerous tumors which cannot be removed entirely through surgery, despite the accompanying adverse effects [3,4]. These drugs and radiations damage the actively proliferating cancer cells and dividing normal cell indiscriminately [5][6][7][8], leading to deterioration in patient health by causing severe weight loss, poor skin conditions and hair loss (alopecia) [9,10]. The adverse effects of chemotherapy and radiotherapy in weakening patients' health and immune system have yet to be resolved despite many years of refinement. In fact, criticisms from patients receiving these treatments discourages many from enrolling as their physical and mental wellbeing are at risk. The percentage of patients refusing to have or continue their chemotherapy is reported to be as high as 3-19% [11]. All these limitations demonstrate the importance of developing novel treatment for cancer which is highly specific and effective in eradicating the cancer cells without affecting the healthy cells. 2 of 25 Stem cells have been studied extensively in the field of regenerative medicine due to their excellent regenerative and anti-inflammatory properties [12]. They have been regarded as a viable solution for many age-related and chronic degenerative illnesses. This development has piqued the interest of utilizing stem cells for other therapeutic applications, including cancer immunotherapy. The development of cell-based immunotherapy can be achieved by establishing cell lines that express tumor selectivity and cytotoxic functions. Several prospective candidates that have been rigorously studied are natural killer (NK) cells, dendritic cells (DC), macrophages and T-lymphocytes [13][14][15][16]. These cells play critical roles in alerting the immune system (e.g., chemotaxis), arming other immune cells (e.g., antibody-dependent and cytokine-dependent activation) and exerting their cytotoxic effect against the cancer cells (e.g., receptor-mediated cell apoptosis). DC and CD4 + T-cells (T-helper cells) are important components of the immune system in that they serve as the moderators of various immune responses [17,18]. They produce molecular signals, e.g., cytokines and chemokines, to guide and activate other immune cells. Conversely, NK cells, macrophages and CD8 + T-cells (cytotoxic T-cells) are effector cells that exert a cytotoxic effect against the cancer cells [19][20][21][22].
The CD8 + T-cell's mechanism of action is through the antibody-dependent cellular cytotoxicity (ADCC). ADCC is initiated by the binding of T-cell receptors (TCR) to the antigen presented by major histocompatibility complex-1 (MHC-1) [23]. NK cells also can eliminate the target cells via ADCC pathway through the cluster of differentiation-16 (CD16) or FcγRIII on its plasma membrane [24,25]. Upon activation, these cells will release cytolytic enzymes (e.g., caspases, granzymes (Gzm) and perforin (PFN)) to damage the target cells. Meanwhile, macrophages elicit antibody-dependent cellular phagocytosis (ADCP), which involves engulfment and degradation of the internalized cells via phagosome acidification [26,27]. Since mutational activities are frequent in cancer cells, antigen or peptide reconfigurations or direct inhibition of cytotoxic receptor (e.g., CA-125 inhibits Fcy-receptor) can disable ADCC/ADCP entirely [27][28][29][30][31]. In the event ADCC and ADCP are inhibited, the immune cells still can eliminate the cancer cells via the TRAIL and Fas signalling pathways. TRAIL and Fas receptors are "death receptors" which belong to the receptor family of tumor-necrosis factors (TNF) that induce programmed cell death when they are cross-linked by its ligands [32][33][34]. In addition, certain immune cells also secrete lytic granules containing Gzm and PFN that induce lysis or apoptosis of target cells via degranulation [35][36][37].
In spite of its potential, the progression of cell-based immunotherapy has been hampered by the limitations that it may cause life-threatening adverse reactions, laborious production procedure, limited efficacy against solid tumors due to insufficient trafficking and homing, and variable efficacy owing to immune cell inactivation by the tumors [38,39]. Recently, purification of cell biologics was made possible by the new innovations in isolation technique. By using these advanced isolation technologies, researchers have isolated the extracellular vesicles (EVs) produced by the immune cells and use them as cancer immunotherapy. In that regard, immune cells have been repurposed as biological manufacturers of these membrane-bound nanovesicles [40]. NK cells-derived EV (NK-EV) is being explored in many studies as it is rich in cytotoxic proteins, cytokines and miRNAs that selectively kill the tumor cells [41]. In the interest of this development, this systematic review was performed to determine the potential of NK-EVs as cancer immunotherapy based on data collected from the in vitro and in vivo experiments. EVs can be classified based on their biogenesis and size into exosomes (EXO), microvesicles (MVs) and apoptotic bodies [42,43]. In this review, NK-EVs are referring to the EXO and MVs produced by the NK cells.

NK-EVs Express Cytotoxicity against Various Human Cancer Cell Lines
A total of 17 studies performed in vitro experiments to examine the efficacy of NK-EVs against a panel of tumor cell lines, and all of them reported positive outcomes ( Table 1). The NK-EVs were found to be effective against a wide range of cancer cell lines, shown in Figure 2 below, such as the brain [44][45][46][47][48][49][50][51], breast [44,45,47,[49][50][51][52][53][54][55], blood [44,48,53,[55][56][57], cervix [57], colon [50,51], liver [45,49,58], lung [57], ovary [51,54], pancreas [59], prostate [51], skin [51,60], stomach/gastric [50,60], and thyroid [45,49]. The dose of NK-EVs used in these studies ranged from low dose, between 0.3−5 µg, to median dose, between 10−25 µg, and high dose, between 40−100 µg. The NK-EVs were found to kill the cancer cells in a time-and dose-dependent manner [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60]. Hence, higher cytotoxicity was recorded when the cancer cell lines were exposed to higher dose of NK-EVs for a longer period. Importantly, the NK-EVs showed selective cytotoxicity towards the cancer cell lines without affecting the viability of the healthy or normal cell lines tested. This evidence clearly showed that NK-EVs exhibited selectivity akin to their parental cells, as reported in the previous literature [61]. NK-EVs were cytotoxic towards both cancer cell lines. PFN, GzmA, GzmB and GNLY were found to work collectively to induce tumor cytotoxicity. NK-EVs were able to enter caspase-dependent and independent pathways, highlighting the flexibility of NK-EVs to access multiple cytotoxic pathways.  Transduced cells produced EVs enriched with BCL-2 siRNAs. Modified NK-EVs showed greater cytotoxicity against the breast cancer cell lines but have no effect on the normal cells. NK-EXOs had a cytotoxicity towards Hep3B compared to HepG2 and Huh7 cells in a dose-dependent but not time-dependent manner. The impressive findings from the in vitro studies merely demonstrated the fundamental anti-cancer potential of NK-EVs without the interference of other biological systems. Thus, the use of mere in vitro data to show the anti-cancer potential of NK-EVs is totally insufficient, as the model does not replicate the physiological complexities that make up a living organism [62]. For more effective translation, a highly defined and complex model of pharmacodynamics (PD) and pharmacokinetics (PK) is needed, such as the one constructed by Bouhaddou et al. (2020) [63]. In term of cancer, the main debacle that has stumped the progression of many medical innovations is the existence of drug resistance, which is very difficult to replicate in vitro [64]. Tumor cells are known to interact with the surrounding cells and tissues, also known as tumor microenvironment (TME), to ensure their survival and the development of drug resistance. The TME provides important support needed by the tumor to thrive, including the secretion of signalling The impressive findings from the in vitro studies merely demonstrated the fundamental anti-cancer potential of NK-EVs without the interference of other biological systems. Thus, the use of mere in vitro data to show the anti-cancer potential of NK-EVs is totally insufficient, as the model does not replicate the physiological complexities that make up a living organism [62]. For more effective translation, a highly defined and complex model of pharmacodynamics (PD) and pharmacokinetics (PK) is needed, such as the one constructed by Bouhaddou et al. (2020) [63]. In term of cancer, the main debacle that has stumped the progression of many medical innovations is the existence of drug resistance, which is very difficult to replicate in vitro [64]. Tumor cells are known to interact with the surrounding cells and tissues, also known as tumor microenvironment (TME), to ensure their survival and the development of drug resistance. The TME provides important support needed by the tumor to thrive, including the secretion of signalling molecules by the proximally located cells, structural support and biochemical signals granted by the altered extracellular matrices (ECM), protection by the immunosuppressive local immune cells, nutrient supply and waste removal by the sprouting blood vessels, and a dynamic microenvironment (e.g., optimal temperature and pH level) that favor cancer cell growth, invasion and metastasis [65][66][67][68].

Tumor Microenvironment (TME): A Physicochemical Barrier against Immunotherapy
The TME is described as an extremely hostile ecosystem consisting of both physical and chemical components, as illustrated in Figure 3 [69,70]. Similar to all other cell types, cancerous cells are no exceptions to the innate ability to secrete EVs. In fact, tumor-derived extracellular vesicles (TD-EVs) are expressed in significant quantities, making them a plausible biological marker and progress indicator for cancer patients [71][72][73]. Cancer cells are also more self-sufficient than regular cells since they are capable of producing and responding to their own growth factors [74]. As demonstrated by El-Fattah Ibrahim et al.
(2019), these tumor-enabling secretions can operate in an autocrine (own cells), paracrine (neighboring cells) and endocrine (distally located cells) manner [75]. TD-EVs were known to alter the surrounding microenvironment, making it hostile for immune cells and other healthy tissue, but their utility and mechanism of action were never fully comprehended. However, they do share a resemblance to anti-inflammatory secretions by supporting cell proliferation and angiogenesis, while inhibiting cell apoptosis, maturation or differentiation, and suppressing the recruitment of inflammatory-responsive cells (e.g., NK cells, macrophages, B and T lymphocytes) [76]. When healthy cells are replaced or destroyed, the surrounding environment is contaminated by the metabolic waste products, inflammasomes from cell lysis, and cell debris. Active glycolysis in malignant cells consumes the surrounding oxygen and releases acidic products (e.g., pyruvate and hydrogen ions) [77]. After depleting the surrounding oxygen, cancer cells will undergo anaerobic glycolysis, which contributes to further acidification via lactic acid production [78]. These manifest into the well-known acidic and hypoxic properties of TME, forming a chemical barrier that limits immune cell penetration. sels, and a dynamic microenvironment (e.g., optimal temperature and pH level) that favor cancer cell growth, invasion and metastasis [65][66][67][68].

Tumor Microenvironment (TME): A Physicochemical Barrier against Immunotherapy
The TME is described as an extremely hostile ecosystem consisting of both physical and chemical components, as illustrated in Figure 3 [69,70]. Similar to all other cell types, cancerous cells are no exceptions to the innate ability to secrete EVs. In fact, tumor-derived extracellular vesicles (TD-EVs) are expressed in significant quantities, making them a plausible biological marker and progress indicator for cancer patients [71][72][73]. Cancer cells are also more self-sufficient than regular cells since they are capable of producing and responding to their own growth factors [74]. As demonstrated by El-Fattah Ibrahim et al.
(2019), these tumor-enabling secretions can operate in an autocrine (own cells), paracrine (neighboring cells) and endocrine (distally located cells) manner [75]. TD-EVs were known to alter the surrounding microenvironment, making it hostile for immune cells and other healthy tissue, but their utility and mechanism of action were never fully comprehended. However, they do share a resemblance to anti-inflammatory secretions by supporting cell proliferation and angiogenesis, while inhibiting cell apoptosis, maturation or differentiation, and suppressing the recruitment of inflammatory-responsive cells (e.g., NK cells, macrophages, B and T lymphocytes) [76]. When healthy cells are replaced or destroyed, the surrounding environment is contaminated by the metabolic waste products, inflammasomes from cell lysis, and cell debris. Active glycolysis in malignant cells consumes the surrounding oxygen and releases acidic products (e.g., pyruvate and hydrogen ions) [77]. After depleting the surrounding oxygen, cancer cells will undergo anaerobic glycolysis, which contributes to further acidification via lactic acid production [78]. These manifest into the well-known acidic and hypoxic properties of TME, forming a chemical barrier that limits immune cell penetration.  What is also seemingly peculiar about TD-EVs is the manipulation of host cells, akin to a virus. TD-EVs are able to influence or reprogram the recipient cells to cooperate with tumors [79]. For example, TD-EVs can falsely trigger the anti-inflammatory machinery of the healthy cells to reduce the TME inflammation. On the other hand, death cells in TME will continuously release inflammatory particles. Hence, TME paradoxically houses both anti-inflammatory particles and pro-inflammatory particles. How these opposing factors co-exists and modulate the TME inflammation is yet to be fully understood. In addition, studies have shown that the circulating inflammatory bodies passively recruit immunoreactive cells to the tumor region [80][81][82][83]. The immune cells recruited to the TME will be modulated by the TD-EVs to deactivate its tumor cell killing function and/or to maintain its status quo via overstimulation of the inhibition:activation signal ratio [84]. At the same time, the aggregation of naïve/resting or inactivated immune cells interlocked by faulty ECMs obstructs any movement and acts as a shield against activated immune cells or medical interventions (e.g., chemotherapy drugs). Thus, the formation of this pseudo-barrier reinforces the physical impenetrability of the tumor, contributing to drug resistance. TME and TD-EVs are known to work in harmony to disrupt NK cell function, thus, reducing the effectiveness of NK cell therapy. The acidic pH, as well as the immunosuppressive myeloid derived suppressor cells' (MDSCs) and M2 macrophages' presence in the TME, suppress the activation of NK cells, thereby compromising their cytotoxicity against a range of tumor cells [85]. Similarly, the TD-EVs were found to impair the function of NK cells through the transfer of multiple immunosuppressive factors (e.g., miRNAs and TGF-β) [86,87]. The existence of physical and chemical barriers in TEM paired with the inhibitory factors from TD-EVs render the NK cells and other immune cells almost entirely incompetent. Cellular release and uptake of EVs are known to increase in acidic environments [88]. Additionally, the pH-related stress also increases the EVs' protein content and surface electrokinetic potential (zeta potential) [89]. The EVs with higher surface charges can bind strongly to the cell membrane, therefore increasing its internalization via receptor or non-receptor endocytosis. The higher EV absorption is not restricted to TD-EVs, but applicable to other EVs as well. This implies the possibility of higher absorption of NK-EVs and other immune cell-derived EVs by the cancer cells.

NK-EVs Show In Vivo Cytotoxicity in Tumor-Bearing Mice
The in vitro to in vivo extrapolation has seen numerous failures due to subpar experimental design and the presence of many limitations that yet to be addressed [62]. Thus, preclinical study needs to be performed to extrapolate animal data to humans despite that it is also unreliable due to species differences. Table 2 describes studies that assess the performance of NK-EV therapy using the in vivo model of tumor-bearing animals and the range of doses per administration [45][46][47]50,53,60,90]. The four studies shown in Figure 4 that performed intra-tumoral (IT) infusion reported significant suppression (p < 0.05) of tumor growth and lowered cancer cell viability in the neoplastic mass [45,53,60,90]. Similar results were reported in the seven studies that performed intravenous (IV) delivery of NK-EVs through the tail vein [45][46][47]49,50,58,90]. In all these studies, NK-EV infusion significantly (p < 0.05) reduced the bioluminescent intensity (BLI) signals and/or physical dimensions of the tumor mass compared to the untreated animals. The study by Zhu et al. (2018) performed a direct comparison between IT and IV administration routes in their animal models [45]. They found that IT administration is more effective (p < 0.05) compared to IV administration in shrinking the tumor mass. Similar results have been reported in previous studies examining the anti-tumor effect of immunotherapies in tumor-bearing mice [91,92]. These findings indicate that the IT route is likely to be more efficacious for NK-EV therapy but the IV route is still viable and may be used in specific situations when the IT route is not accessible.   The SYRCLE's risk of bias tool adopted from Hooijman 2014 was used to assess the risk of bias of the selected studies ( Figure 5) [93]. Generally, most of the elements evaluated showed an unknown risk of bias. While most of the studies demonstrated low risk of bias for some of the elements investigated, a few studies were found to have high risk. Among the nine studies reviewed, five studies (55.6%) explicitly mentioned randomization [45][46][47]50,58] but three studies (33.3%) did not [49,53,60]. A single study (11.1%) was deemed high-risk due to undisclosed sample size and allocation method [90]. The baseline was presented prior to treatment in 6 studies (66.7%) but ambiguous in the remaining three studies (33.3%). None of the studies disclosed information for allocation concealment, random housing, blinding of interventions or caregivers, random outcome assessment, and blinding of outcome assessment. Nearly all studies (n = 8, 88.9%) have unknown risk of bias for selective outcome reporting bias, with one study has high-risk of bias [50]. Five studies (55.5%) reported low risk of attrition bias and three (33.3%) had an unclear risk, with one (11.1%) having high-risk [50]. The unclear risk is mainly attributed to the inconsistency in the sample size of the study's outcomes with the number of animals allocated per group. Failure to disclosure the number of animals (alive or dead) excluded from the analysis or parameter conducted were also took into account. All studies were at low risk of "other sources of bias" by disclosure of ethics approval, potential conflict of interest, or the collaborating parties and their roles. The SYRCLE's risk of bias tool adopted from Hooijman 2014 was used to assess the risk of bias of the selected studies ( Figure 5) [93]. Generally, most of the elements evaluated showed an unknown risk of bias. While most of the studies demonstrated low risk of bias for some of the elements investigated, a few studies were found to have high risk. Among the nine studies reviewed, five studies (55.6%) explicitly mentioned randomization [45][46][47]50,58] but three studies (33.3%) did not [49,53,60]. A single study (11.1%) was deemed high-risk due to undisclosed sample size and allocation method [90]. The baseline was presented prior to treatment in 6 studies (66.7%) but ambiguous in the remaining three studies (33.3%). None of the studies disclosed information for allocation concealment, random housing, blinding of interventions or caregivers, random outcome assessment, and blinding of outcome assessment. Nearly all studies (n = 8, 88.9%) have unknown risk of bias for selective outcome reporting bias, with one study has high-risk of bias [50]. Five studies (55.5%) reported low risk of attrition bias and three (33.3%) had an unclear risk, with one (11.1%) having high-risk [50]. The unclear risk is mainly attributed to the inconsistency in the sample size of the study's outcomes with the number of animals allocated per group. Failure to disclosure the number of animals (alive or dead) excluded from the analysis or parameter conducted were also took into account. All studies were at low risk of "other sources of bias" by disclosure of ethics approval, potential conflict of interest, or the collaborating parties and their roles.

NK-EVs Overcome the Chemical and Physical Barriers of TME
NK-EVs inherit the components and functions of NK cells to exert cytotoxicity against tumor cells in vitro and in vivo. NK-EVs not only express tumor-targeting ability but also possess homing and migratory properties via chemotaxis [45][46][47]50,58]. As depicted in Figure 6, NK-EVs' framework is significantly more compact (30-200 nm) and they are also more biostable than NK cells [94,95]. These properties enable them to have improved infiltration and survival in the hostile TME. It has been demonstrated that NK-EVs express NKG2D, FasL and TRAIL just like the parent cells for receptor-mediated apoptosis. Lytic proteins such as PFN and Gzm also exist ubiquitously in NK-EVs to initiate caspase-dependent apoptosis pathway. All these functions show that the simple configuration of NK-EVs does not compromise their capability to eliminate cancer cells  [45][46][47]49,50,53,60,90]. Symbols: yellow "?" indicates unclear risk; green "+" indicates low risk; and red "-" indicates high risk of bias.

NK-EVs Overcome the Chemical and Physical Barriers of TME
NK-EVs inherit the components and functions of NK cells to exert cytotoxicity against tumor cells in vitro and in vivo. NK-EVs not only express tumor-targeting ability but also possess homing and migratory properties via chemotaxis [45][46][47]50,58]. As depicted in Figure 6, NK-EVs' framework is significantly more compact (30-200 nm) and they are also more biostable than NK cells [94,95]. These properties enable them to have improved infiltration and survival in the hostile TME. It has been demonstrated that NK-EVs express NKG2D, FasL and TRAIL just like the parent cells for receptor-mediated apoptosis. Lytic proteins such as PFN and Gzm also exist ubiquitously in NK-EVs to initiate caspase-dependent apoptosis pathway. All these functions show that the simple configuration of NK-EVs does not compromise their capability to eliminate cancer cells through the multiple cytotoxic pathways utilized by its parent cells. Instead, the NK-EVs might be more effective without the interference of the TME and TD-EVs that halt the production of PFN and Gzm in NK cells. It is believed that the anti-cancer potency of NK-EVs is mainly attributed by these killer proteins. However, the concentration and ratio of NK-EVs in relative to TD-EVs in the TME will likely determine if they will be able to exert anti-tumor effects. It is highly possible that the level of inhibitory signals induced by TD-EVs may overwhelm the activating signals of NK-EVs to prevent any cytotoxic action [84]. This means that the dose and delivery method of NK-EVs in vivo need to be critically evaluated to ensure their high bioavailability in the tumor in order to exert their cytotoxic function.

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NK-EVs in relative to TD-EVs in the TME will likely determine if they will be able to exert anti-tumor effects. It is highly possible that the level of inhibitory signals induced by TD-EVs may overwhelm the activating signals of NK-EVs to prevent any cytotoxic action [84]. This means that the dose and delivery method of NK-EVs in vivo need to be critically evaluated to ensure their high bioavailability in the tumor in order to exert their cytotoxic function.

NK-EVs Are More Clinically Applicable than NK Cells
Results from the in vitro and in vivo experiments demonstrated that NK-EVs are an effective and safe immunotherapy. However, there are several foreseeable issues that need to be addressed in preparing this therapy for future clinical trials and registration. To begin, the functions of NK cells are distinguishable by their cell activation status [24,96,97]. Generally, both naïve and activated NK cells can secrete NK-EVs [98]. However, the activated NK cells are preferable for EV isolation due to their high cell number

NK-EVs Are More Clinically Applicable than NK Cells
Results from the in vitro and in vivo experiments demonstrated that NK-EVs are an effective and safe immunotherapy. However, there are several foreseeable issues that need to be addressed in preparing this therapy for future clinical trials and registration. To begin, the functions of NK cells are distinguishable by their cell activation status [24,96,97]. Generally, both naïve and activated NK cells can secrete NK-EVs [98]. However, the activated NK cells are preferable for EV isolation due to their high cell number in in vitro culture. It has been reported that the NK-EVs derived from inactivated NK cells have lower amounts of cytotoxic proteins [46]. In a separate study, the authors found that EVs secreted by the NK92 cell line contain fewer cytotoxic proteins compared to the EVs secreted by in vitro-expanded NK cells [48]. Additionally, Shoae-Hassani et al. (2017) reported that NK cells exposed to neuroblastoma cells produced NK-EVs with higher cytotoxicity towards the tumor cells in vitro and in vivo compared to the NK-EVs derived from naïve NK cells [99]. Priming of NK cells with IL-15 was also reported to increase the cytotoxicity of NK-EVs [49]. These observations show that NK-EVs are highly heterogeneous depending on the cellular origin, culture environment and physiological status. Therefore, it is important to conduct experiments to the optimize the NK cell culture protocol in order to harvest NK-EVs with potent therapeutic potential. Importantly, the optimization should also consider increasing the yield of NK-EVs in order to reduce the cost.
Unlike the stem cell, donor-derived NK cells have variable and limited expansion potential in vitro [100]. Thus, efforts have been made to prepare immortalized NK cell lines, such as the NK92-MI from the American Type Culture Collection (ATCC, USA). From Table 1 [48,51]. Cryopreservation is widely used for long-term storage of NK cells. However, the cryopreserved NK cells have relatively poor viability albeit different freezing medium have been tested. Many studies have reported cell viability less than 10% after revival [101]. More alarming is that the survived cells ceased proliferating and failed to exert any cytotoxic functions [102]. Failure of cell cryopreservation will definitely hamper the clinical translation of NK-EV therapy as it will limit the potential of producing them on a large scale and consistently. At the moment, new donor-derived NK cells are used to prepare the NK-EVs, which leads to high batch-to-batch variation. Furthermore, it will also increase the production cost and lead time.
Manual cell culture protocol using tissue culture flask is prone to contamination, technical errors and high batch-to-batch variation [103,104]. To overcome these limitations, efforts should be made to shift the NK cell expansion to a bioreactor platform which is highly automated, allows more control over the culture environment (e.g., glucose concentration, oxygen concentration and pH) and requires less manpower [105]. Usage of a highly efficient bioreactor system might also reduce the cost of production [44]. Not least, it is important is to optimize the EV isolation protocol, which should allow quick and reliable isolation of EV subpopulation.
Last but not least, long-term storage and off-the-shelf availability are highly desirable for NK-EV therapy. To achieve this, researchers need to device a storage condition that can preserve EV stability over a long period of time. As an acellular product, NK-EVs can be kept chill (4 • C) for immediate use or frozen (−20 • C or −80 • C) up to 12 months with acceptable loss of proteins and RNAs [106][107][108][109][110]. More importantly, the NK-EVs retain their anti-tumor effect. For easier storage and transportation, NK-EVs could be lyophilized. Even though lyophilization has been widely explored for storage of stem cell-derived EVs, it is still unknown whether lyophilization can be used to preserve the NK-EVs.

Defining Nomenclature, Isolation Technique, QA/QC Methods and Biomarkers for NK-EVs
In the very beginning, EVs were first thought as a mechanism of disposing cellular waste products [111]. Later, studies found that these minute vesicles are rich in lipids, proteins, nucleic materials (e.g., DNA, RNA, mRNA, miRNA) and other biologically active molecules [112]. On their surface, they express different molecules and proteins adopted from parent cell membrane during the budding or exocytosis process. These physical hallmarks contribute to the diverse functions and heterogeneous properties of EV molecules. Size is commonly used to classify EVs [113]. The largest EVs are the apoptotic bodies (1000-5000 nm), followed by microvesicles (50-1000 nm), with exosomes (30-150 nm) being the smallest [114]. The term "exosome" is often incorrectly used interchangeably with "extracellular vesicle" to describe the small EV preparation [42]. This is because most of the studies isolate the EVs based on their size instead of the specific phenotype that represented their unique biogenesis pathway. Every EV subset has a specific protein phenotype and molecular content. Thus, the Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018 guidelines have recommended the nomenclature "extracellular vesicles" to describe the small membrane-bound particles secreted by the cells. It is crucial to establish a uniform and specific standard for appropriate evaluation of different NK-EV preparations as it would allow researchers to determine the strengths and weaknesses of any modifications or novelties introduced.

Future Considerations of NK-EVs as a Tool for Immunotherapy
They are safer alternatives with potentially fewer procedural complications to the highrisk populations such as infants and elderly. In contrast, surgical treatment can lead to many serious complications, including induced hemorrhage, post-operative sepsis and extended rehabilitation [121,122]. Selection of administration route or delivery technique is critical to ensure the safety and efficacy of a therapy [123]. Although IT administration would eliminate any concerns over the homing and migration ability of NK-EVs, the IV administration will be especially useful for regions with poor access or penetrability (e.g., brain) [124]. In actuality, a larger dose is typically needed for IV route to compensate for the accumulated loss of drugs due to unwanted distribution at the other tissues as well as metabolism and excretion primary at the liver and kidney [125][126][127]. Nonetheless, the continual IV administration of NK-EVs after the tumor elimination could be favourable to ensure complete clearing of tumor cells to minimize the risk of relapse [128,129]. To date, it is still unclear on the optimal dosage regime of NK-EVs. More research is needed to determine the ideal frequency, dosage and interval of NK-EV treatment.
Efforts are also needed to improve the efficacy of NK-EV production in order to reduce the cost. Owing to its niche role, NK cells have high metabolic activity and need specific cytokines to stimulate its proliferation. The commonly used cytokines to expand NK cells are IL-2, IL-15, IL-18, and IL-21. The cytokines can be provided by the peripheral blood mononuclear cells (PBMCs) that serve as the feeder cells [130]. Alternatively, the culture medium can be supplemented with commercially available human recombinant cytokines to maintain the NK cell culture. Although the use of recombinant cytokines is simpler as no purification step is needed to remove the contaminating PBMCs and safer as it is free from PBMC contamination, the cost of recombinant cytokines is very high. The high concentration and cost of recombinant cytokines will significantly escalate the production cost. On top of that, the requirement of using GMP-grade or clinicalgrade raw materials and consumables, skilled manpower, stringent quality assurance and quality control (QA/QC), expansive equipment for scalable production, and the use of a cGMP-certified production facility will escalate the treatment cost to unaffordable scales for majority of the patients [131]. This, in turn, will affect the clinical translation and commercial viability of NK-EV therapy.

Conclusions
NK-EVs show promising tumor cytotoxicity with no adverse or side effects in vitro and in vivo. NK-EVs are not cytotoxic to the normal or healthy cells and can increase the life span of tumor-bearing animals. Thus, this novel therapy has great clinical potential. In addition, NK-EVs can circumvent some of the limitations of NK cell therapy, such as impediment of NK cell functionality by the TME and TD-EVs. In fact, NK-EVs would see potential benefits due to the increased cellular uptake in the TME. Although IT administration is superior, the IV administration will be meaningful for "difficult-to-reach" tumors and high-risk populations. Additionally, NK-EVs can be readily available off-the-shelf as they are more stable and easier to store compared to NK cells. Nonetheless, NK-EV therapy and its production procedure need further investigation to improve its safety and efficacy as well as to increase the yield and lower the production cost.